By crosslinking IgE bound to the high affinity IgE receptor, FceRI, allergens initiate a complex sequence of events leading to release of mast cell inflammatory mediators. We have used a combination of high resolution microscopy techniques and lipidomics to show that IgE receptors localize to distinct microdomains in the plasma membrane during signaling and receptor internalization. Our central hypothesis is that this membrane organization is critical to the regulation of mast cell responses. Based upon recent evidence, we propose that membrane proteins are concentrated in "protein islands," protein and cholesterol-rich domains separated by expanses of mostly lipid. IgE receptors are transiently confined within these small, heterogenous islands, providing one mechanism for receptor clustering observed by EM. Molecular segregation can occur within islands, facilitating signal transduction and at least two pathways of endocytosis. Cytoskeletal elements, including actin-based corrals and cortical cytoskeleton networks, restrict receptor mobility and may also serve as tethers for protein islands. In this proposal, we plan to test several hypotheses related to our model. In Aim 1, we will develop new monovalent probes for plasma membrane lipids. We will use both transmission and X- ray spectral electron microscopy to map the distributions of tagged lipids on membrane sheets. We will also use fluorescent cholesterol derivatives to determine if cholesterol-rich regions in the outer and the inner leaflet of the membrane bilayers overlap and to study cholesterol diffusion relative to FceRI. Results in the rat basophilic leukemia (RBL-2H3) cell line will be confirmed in BMMCs. In Aim 2, we will test the hypothesis that receptor diffusion and antigen-stimulated signal initiation is modified by part-time residency in protein islands and by elements of the cortical cytoskeleton. We have documented the expression of 2 a spectrin and 3 [unreadable] spectrin isoforms in RBL-2H3 cells. We will use RNA interference and transient transfection methods to disrupt the spectrin cortical network. Single particle tracking (SPT) with multiple colors of monomeric IgE-quantum dot probes will reveal consequences of this disruption for receptor mobility and clustering behavior. Stochastic modeling methods will be used to determine a best fit model for the anomalous diffusion and clustering of IgE receptors, comparing contributions of 0.5-1 micron corrals, smaller cortical cytoskeletal subdomains and protein islands. Assays for calcium flux, secretory response and cytokine production will correlate functional responses with receptor immobilization, clustering and desensitization. In Aim 3, we will test the hypothesis that both clathrin-dependent and clathrin-independent endocytic pathways mediate internalization of receptors from primary signaling domains. We will isolate CLICs (clathrin-independent vesicular carriers) responsible for FceRI endocytosis in the absence of clathrin. We will use a combination of proteomics, live cell and electron microscopy, pharmacologic, and molecular biology approaches to identify and characterize molecular regulators of non-clathrin-mediated endocytosis. A particular focus in this aim is the small GTPase, Arf6. Results of these analyses will contribute important new insight specifically into the molecular events that initiate allergic inflammation and more generally into the nature and assembly of membrane microdomains controlling signal transduction and membrane trafficking in immune cells. Public Health Relevance: Allergic diseases are on the rise in the United States. Mast cells and basophils are the principal mediators of the allergic response, through activation of the high affinity IgE receptor, FceRI. When these receptors are crosslinked by polyvalent allergens, the cells release inflammatory mediators such as histamine and leukotrienes. We use sophisticated microscopy techniques and analytical approaches to study the behavior of IgE receptors in the mast cell membrane. We have discovered that the plasma membrane has a rich topography, that we believe is critical to the regulation of mast cell responses. The landscape of the mast cell membrane undergoes dramatic change during cell activation and receptors come together into large clusters in order to signal to the cell interior. The signaling process is limited in part by uptake of the receptors into the cell, a process referred to as endocytosis. In this proposal, we will study the dynamic behavior of receptors in live cell membranes using brightly fluorescent nanoprobes called quantum dots. We will also use our innovative electron microscopy methods to map the distributions of lipids, receptors and other proteins across the mast cell landscape. We will gain important insight into the mechanisms that drive receptor endocytosis. Our work is applicable to other cell types, particularly other cells of the immune system. Results of these analyses will contribute important new insight into the nature and assembly of membrane microdomains controlling immune cell signaling.